Studies of binding of cardiac glycosides to NaK-ATPase suggest that they are bound to the enzyme at two sites, one specific for the steroid moiety and the other for the sugar. Complex formation between the enzyme and glycoside requires the presence of either Na ion, Mg 2 ion, and ATP or Mg 2 ion and Pi as ligands. Our results indicated that for the steroid (aglycone) binding, these ligands were also required and the concentration of the above ligands did not influence the dissociation constant of digoxigenin but changed the number of binding sites. These results suggest that the binding of cardiac aglycones does not represent the usual reversible reaction but is due to various enzyme conformation(s), the formation of which is dependent on the particular ligand system employed. The interactions between NaK-ATPase and cardiac aglycone in the presence of various ligands will be studied by measuring 1) the association rate constants and 2) the dissociation rate constants. For these studies, rapid mixing techniques will be employed and the formation of 32P-labelled phosphoprotein can be used to evaluate the extent of interaction obtained. This approach will define the specific transient intermediate necessary to bind various cardiac aglycones. Addition to such direct approaches, Species-dependency of sensitivity to cardiotonic steroid, the differences in the binding of cardiac aglycones and glycosides to NaK-ATPase preparations from hearts of various species (beef, rat and frog) having different sensitivities to cardiac drugs will be studied. These various approaches will contribute to our understanding of the action of cardiotonic steroids.